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WO2019113085A1 - Systèmes de détection électrochimique et composants de ceux-ci - Google Patents

Systèmes de détection électrochimique et composants de ceux-ci Download PDF

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Publication number
WO2019113085A1
WO2019113085A1 PCT/US2018/063866 US2018063866W WO2019113085A1 WO 2019113085 A1 WO2019113085 A1 WO 2019113085A1 US 2018063866 W US2018063866 W US 2018063866W WO 2019113085 A1 WO2019113085 A1 WO 2019113085A1
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formula
tethered
polymer
subunit
aryl
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Logan Earl GARNER
Luke Satish Kumar THEOGARAJAN
William Van Antwerp
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Laxmi Therapeutic Devices Inc
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Laxmi Therapeutic Devices Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue
    • A61B5/14507—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue specially adapted for measuring characteristics of body fluids other than blood
    • A61B5/1451—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue specially adapted for measuring characteristics of body fluids other than blood for interstitial fluid
    • A61B5/14514—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue specially adapted for measuring characteristics of body fluids other than blood for interstitial fluid using means for aiding extraction of interstitial fluid, e.g. microneedles or suction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/145—Hydrogels or hydrocolloids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F120/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
    • C08F120/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F120/52—Amides or imides
    • C08F120/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F120/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L33/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Compositions of derivatives of such polymers
    • C08L33/24—Homopolymers or copolymers of amides or imides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G01N27/3273—Devices therefor, e.g. test element readers, circuitry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/4166—Systems measuring a particular property of an electrolyte
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L2203/00—Applications
    • C08L2203/02—Applications for biomedical use
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)

Definitions

  • the invention is directed to electrochemical detection systems, components thereof, materials and methods for making the systems and the components thereof, and methods of detecting analytes with the systems.
  • the quantitative determination of analytes in bodily fluids is important for the diagnoses and maintenance of certain physiological abnormalities. For example, lactate, cholesterol, and bilirubin should be monitored in certain individuals. In particular, it is important that diabetic individuals frequently check the glucose level in their body fluids to regulate the glucose intake in their diets. The results of such tests can be used to determine what, if any, insulin or other medication needs to be administered.
  • Electrochemical systems have been used for detecting analytes in a sample. These systems, however, have difficulty detecting analytes in small volumes, such as in the nanoliter and picoliter range. Materials suitable for detecting analytes in small sample volumes are needed.
  • An exemplary aspect of the invention includes an electrochemical detection system based on functional hydrogel materials containing immobilized oxidoreductase enzymes.
  • the system can be used for detecting analytes such as glucose.
  • the general detection system architecture includes a two- or three-electrode electrochemical cell functionalized with the enzyme-containing hydrogel material. Sensing is based upon direct or indirect measurement of electrons from enzymatic analyte oxidation.
  • hydrogel material which serves to immobilize the enzyme component in close proximity to the electrodes, regulate mass transport rates of analytes, and, in some versions of the invention, mediate transport of electrons from oxidoreductase enzyme to electrode.
  • the features of the hydrogel materials result in a detection system that is amenable to multiple detection modes that provide the ability to tune operating voltages, maximize sensitivity, and minimize background signal.
  • the disclosed detection system drastically reduces the minimum sample volume to the nanoliter and even picoliter range, which is well below the present state of the art.
  • Another exemplary aspect of the invention includes versatile hydrogel and redox hydrogel functional materials containing oxidoreductase enzymes that can be used for next generation electrochemical enzymatic biosensors.
  • the materials include hydrophilic polymers, hydrogels, and redox hydrogels.
  • the polymers can be prepared from acrylamide/methacrylamide and modified acrylamide/methacrylamide co-monomers equipped with pendants bearing amine and cationic ammonium functional groups.
  • the amine and ammonium groups act as sites for functionalization (/. ⁇ ? ., tuning of materials to achieve desired traits or sites that facilitate desired processes/modifications) and/or that undergo desired or favorable interactions.
  • the polymers include chains of hydrophilic repeating units decorated with the amine, cationic ammonium or other functional groups that allow the polymers to be modified for a wide range of specific applications. Long and flexible pendants facilitate favorable reactions and interactions by endowing the amine and ammonium groups with increased amplitude of motion relative to such groups confined in close proximity to the polymer backbone.
  • the multifunctional hydrogel materials disclosed herein address the materials-based needs of next generation electrochemical enzymatic sensing systems.
  • the materials disclosed herein have a high degree of versatility, which provides functionality for addressing the majority of challenges associated with the development of next generation sensing technologies.
  • Another exemplary aspect of the invention includes acrylamide-, alkylarylamide-, acrylate-, and alkylacrylate-based monomer building blocks equipped with pendant oligo(ethylene glycol) chains bearing terminal nitrogen-containing functional groups, and methods of preparing same.
  • the monomers are water-soluble monomers and can serve as building blocks of hydrophilic polymer-based functional materials for the hydrogels and enzymatic biosensing systems described above.
  • the monomers can be prepared in very few synthetic steps using mild conditions and can be readily equipped with a wide range of functional groups.
  • the monomers can be functionalized with a variety of linking groups prior to undergoing polymerization.
  • Another exemplary aspect of the invention includes redox mediators, including electron shuttles, that can be used in the systems and detection methods of the invention.
  • Another exemplary aspect of the invention includes methods of detecting an analyte, such as glucose with the systems described herein.
  • Other exemplary aspects of the invention include methods of making the systems, polymers, monomers, and redox mediators of the invention.
  • FIGS. 1A-1D show generalized structures of exemplary monomer building blocks of polymers of the invention.
  • R 1 in each instance can independently be H, alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof.
  • R 2 in each instance can independently N or O.
  • R 3 in each instance can independently be, when R 2 is N: H; alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof; or a nitrogen protecting group.
  • R 3 is absent when R 2 is O.
  • R 4 in each instance can be a spacer arm.
  • R 6 , R 7 , and R 8 in each instance can independently be H; alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof; a nitrogen protecting group; a tethered polypeptide; a tethered redox mediator; or a linking arm, with the proviso that at least one of R 6 , R 7 , and R 8 may be absent.
  • FIG. 2A shows schemes for preparing monomers having various tethered linking groups prior to polymer polymerization.
  • the various R groups can be as defined for FIGS. 1A-D.
  • FIG. 2B shows various linking groups that can constitute R 11 in FIG. 2A.
  • R in FIG. 2B can be H; alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof.
  • FIGS. 3A-3G show various exemplary monomers that can be polymerized to generate polymers of the invention.
  • the depicted counterions can be replaced with any counterion, such as chloride, bromide, etc.
  • FIGS. 3F tetra(ethylene glycol) diacrylate (TEGDA)
  • TEGDA tetra(ethylene glycol) diacrylate
  • 3G polyethylene glycol dimethylacrylamide
  • Synthesis schemes for generating the monomers depicted in FIGS. 3D and 3E are shown below in FIGS. 4A and 4B, respectively. Synthesis schemes for generating the monomers shown in FIGS. 3A-3C are described elsewhere herein.
  • FIGS. 4A-4B show schemes for synthesizing the monomers of FIGS. 3D and 3E, respectively.
  • FIGS. 5A-5I show base structures (no tethering functionality or substitutions shown) of exemplary electron shuttles.
  • FIG. 5A depicts a ferrocene electron shuttle.
  • FIG. 5B depicts a 1, 4-naphthoquinone electron shuttle.
  • FIG. 5C depicts an anthroquinone electron shuttle.
  • FIG. 5D depicts a tetrathiafulvalene electron shuttle.
  • FIG. 5F depicts a tetracyanoquinodimethane electron shuttle.
  • FIG. 5F depicts a l-methylphanazine electron shuttle.
  • FIG. 5G depicts a 2,6-dichlorophenolindophenol electron shuttle.
  • FIG. 5H depicts an indigo carmine electron shuttle.
  • FIG. 51 depicts a methylene blue electron shuttle.
  • FIG. 6 shows examples of soluble and tethered 1, 2-naphthoquinones as exemplary electron shuttles.
  • FIGS. 7A and 7B show schemes for synthesizing exemplary 1, 2-naphthoquinones for use as electron shuttles.
  • R 16 can be H, alkyl, or perfluoroalkyl.
  • R 17 can be H, alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof; a tethered polymer; a tethered monomer; or a linking arm.
  • R 17 can be tethered to a polymer monomer or a polymer via any one of R 6 , R 7 , and R 8 in the polymers and monomers provided herein.
  • FIG. 8 shows an exemplary reaction for generating a 1, 2-naphthoquinone electron shuttle comprising a tether with an unreacted linking group.
  • FIGS. 9A-9D show structures of exemplary tethering agents.
  • FIG. 9A depicts glutaraldehyde as a tethering agent.
  • FIG. 9B depicts a tethering agent including N- hydroxysuccinimide and maleimide terminal linking groups and an alkyl chain spacer arm.
  • FIG. 9C depicts a tethering agent including A-hydroxysuccinimide and maleimide terminal linking groups and a polyethylene glycol spacer arm.
  • FIG. 9D depicts a tethering agent including diglycidyl terminal linking groups and a polyethylene glycol spacer arm. In each instance, n can be 0-20 or more.
  • FIG. 10 shows generalized structures of exemplary amine-reactive functional groups.
  • FIGS. 11A-11C show exemplary corresponding linking groups.
  • FIGS. 11A-11C show exemplary corresponding linking groups.
  • FIG. 12A shows a schematic cartoon representation for (A) enzyme tethering post-polymerization; (B) enzyme tethering and electron shuttle installation post polymerization; and (C) enzyme tethering, electron shuttle installation, and cross-linking (via polymer backbone-to-polymer backbone tethers) to form a hydrogel starting from a mixture of enzyme and linear polymer.
  • FIG. 12B shows a schematic cartoon representation for electron shuttle installation, enzyme tethering, and cross-linking (via polymer backbone-to-polymer backbone tethers) in a single step.
  • FIG. 12C shows a schematic cartoon representation for electron shuttle installation, enzyme tethering, and cross-linking (via polymer backbone-to-polymer backbone tethers) in sequential steps.
  • FIGS. 13A-13G show various reactants and products for tethering electron shuttles to amino end groups of polymer pendant groups using various electron shuttles configured with tethering arms (e.g., spacer arms with a unreacted terminal linking group).
  • FIG. 13A shows a cross-linked polymer with primary and quaternary amine- tethered pendants generated using monomers such as those shown in FIGS. 1A-1D that can be used as a reactant for tethering electron shuttles.
  • FIG. 13B shows a ferrocene species (an electron shuttle) with a conjugated tethering arm comprising an epoxide as an unreacted terminal reactive group.
  • FIG. 13C shows a polymer product from reacting the polymer of FIG.
  • FIG. 13A shows a generalized structure of a naphthoquinone electron shuttle with a conjugated tethering arm.
  • FIG. 13E shows an electron shuttle-tethered polymer resulting from reacting the electron shuttle of FIG. 13D with the polymer of FIG. 13A.
  • each R 11 is a linking group, wherein the R 11 moieties in FIG. 13D are one each of a unreacted terminal linking group and a reacted linking group, and R 11 moieties in FIG. 13E are both reacted linking groups, and R 4 is a spacer arm.
  • the sulfonamide (the -SCFNRN subunit) bound to R 11 in FIGS. 13D and 13E can be replaced with sulfhydryl, ester, substituted aryl (such as fluorinated benzene ring), heterocycles, amides, alkylene-amide, or other functional group that is stable, preferably electron withdrawing, and serves as a bridge or connection to a linking group on a tethering agent, or may be absent.
  • FIG. 13F shows a piperazine-containing naphthoquinone electron shuttle with a conjugated tethering arm.
  • FIG. 13G depicts a naphthquinone-tethered polymer product resulting from reacting the electron shuttle of FIG. 13F with a pendant amine on a polymer.
  • each instance of m, n, o, p, q, and r independently represents a positive integer. In some versions, r can be 1-10.
  • FIG. 14A shows an exemplary structural representation for electron shuttle installation, enzyme tethering, and cross-linking (via polymer backbone-to-polymer backbone tethers) in a single step with polymers made with the monomers of FIGS. 3A and 3C and acrylamide.
  • FIG. 14B shows an exemplary structural representation for electron shuttle installation, enzyme tethering, and cross-linking (via polymer backbone-to-polymer backbone tethers) in a single step with polymers made with the monomer of FIG. 3D and acrylamide.
  • FIGS. 15A-15Q show various exemplary reactions for independently installing electron shuttles and cross-linking polymer chains post polymerization on an exemplary starting polymer chain using a variety of different orthogonal chemistries.
  • FIG. 15A shows coupling of an exemplary 2-naphthoquinone electron shuttle to an exemplary linear polymer using NHS-ester based coupling.
  • FIG. 15B shows the preparation the exemplary 2-naphthoquinone electron shuttles used in the NHS-ester based coupling of FIG. 15A and the carbodiimide COOH-amine coupling of FIG. 15L.
  • FIG. 15C shows a first step in tetrazole photo-click cross-linking of the polymer product of FIG. 15A.
  • FIG. 15A shows coupling of an exemplary 2-naphthoquinone electron shuttle to an exemplary linear polymer using NHS-ester based coupling.
  • FIG. 15B shows the preparation the exemplary 2-naphthoquinone
  • FIG. 15D shows a second step in tetrazole photo-click cross-linking of the polymer product of FIG. 15A.
  • FIG. 15E shows a first step of coupling of an exemplary ALO-containing 2- naphthoquinone electron shuttle or an exemplary DIMAC-containing 2-naphthoquinone electron shuttle to an exemplary linear polymer using Cu-free azide-cycloalkyne click chemistry.
  • FIG. 15F shows a second step of coupling of an exemplary ALO-containing 2- naphthoquinone electron shuttle or an exemplary DIMAC-containing 2-naphthoquinone electron shuttle to an exemplary linear polymer using Cu-free azide-cycloalkyne click chemistry.
  • FIG. 15E shows a first step of coupling of an exemplary ALO-containing 2- naphthoquinone electron shuttle or an exemplary DIMAC-containing 2-naphthoquinone electron shuttle to an exemplary linear polymer using Cu-free azide-cyclo
  • FIG. 15G shows the preparation of the exemplary ALO-containing electron shuttle used in the coupling of FIG. 15F.
  • FIG. 15H shows the preparation of the exemplary DIMAC-containing electron shuttle used in the coupling of FIG. 15F.
  • FIG. 151 shows a first step in tetrazole photo-click cross-linking of the polymer product of FIG. 15F.
  • FIG. 15J shows a second step in tetrazole photo-click cross-linking of the polymer product of FIG. 15F.
  • FIG. 15K shows epoxide based cross-linking of the polymer product of FIG. 15F.
  • FIG. 15L shows coupling of an exemplary 2-naphthoquinone electron shuttle to an exemplary linear polymer using carbodiimide COOH-amine coupling.
  • FIG. 15M shows NHS-ester based cross-linking of the polymer product of FIG. 15L.
  • FIG. 15N shows a first step in diazirine photochemical cross-linking of the polymer product of FIG. 15L.
  • FIG. 150 shows a second step in diazirine photochemical cross-linking of the polymer product of FIG. 15L.
  • FIG. 15P shows a first step in aryl azide photochemical cross-linking of the polymer product of FIG. 15L.
  • FIG. 15Q shows a second step in aryl azide photochemical cross-linking of the polymer product of FIG. 15L.
  • FIGS. 16A and 16B show cartoon illustrations of detection system operation based on (FIG. 16 A) direct hydrogen peroxide detection and (FIG. 16B) electron shuttle- based detection with tethered electron shuttles.
  • FIGS. 17A-17D shows cartoon illustrations of four exemplary electrochemical detection modes, including (FIG. 17A) direct hydrogen peroxide detection, (FIG. 17B) electron shuttle-based detection, (FIG. 17C) mediated hydrogen peroxide oxidation detection, (FIG. 17D), mediated hydrogen peroxide reduction detection.
  • FIGS. 17A-17D shows cartoon illustrations of four exemplary electrochemical detection modes, including (FIG. 17A) direct hydrogen peroxide detection, (FIG. 17B) electron shuttle-based detection, (FIG. 17C) mediated hydrogen peroxide oxidation detection, (FIG. 17D), mediated hydrogen peroxide reduction detection.
  • Depicted steps include: (1) glucose binding and enzyme-catalyzed oxidation resulting in the reduced form of enzyme; (2) oxidation of reduced form of enzyme by oxygen to form hydrogen peroxide; (3) diffusion of hydrogen peroxide; (4) electrochemical oxidation of hydrogen peroxide at the working electrode; (2B) oxidation of reduced form of enzyme by oxidized form of electron shuttle resulting in reduced form of electron shuttle; (3B) transport of electrons to electrode via tethered or dissolved electron shuttles; (4B) oxidation of reduced form of electron shuttles at working electrode; (4C) mediated oxidation of hydrogen peroxide; (5C) oxidation of reduced form of mediator at working electrode; (4D) mediated reduction of hydrogen peroxide; and (5D) reduction of oxidized form of mediator at working electrode.
  • FIGS. 18A-18C show exemplary redox mediators capable of undergoing redox reactions with hydrogen peroxide.
  • FIG. 18A shows a cobalt-phthalocyanine redox mediator.
  • FIG. 18B shows a ferrocyanide redox mediator.
  • FIG. 18C shows a Prussian blue redox mediator.
  • FIG. 19 shows a cartoon illustration of a dual detection mode system in which the operating potential is such that both hydrogen peroxide and the reduced form of the electron shuttle can be oxidized at the working electrode.
  • This detection mode permits collecting electrons from enzymatic glucose oxidation in the presence or absence of oxygen and is therefore less susceptible to problems caused by variations in oxygen concentration.
  • Depicted steps include: (1) glucose binding and enzyme-catalyzed oxidation resulting in the reduced form of enzyme; (2 A) oxidation of reduced form of enzyme by oxidized form of electron shuttle resulting in reduced form of electron shuttle; (3 A) transport of electrons to electrode via tethered or dissolved electron shuttles; (4 A) oxidation of reduced form of electron shuttles at working electrode; (2B) oxidation of reduced form of enzyme by oxygen to form hydrogen peroxide; (3B) diffusion of hydrogen peroxide; and (4B) electrochemical oxidation of hydrogen peroxide at the working electrode.
  • FIG. 20 shows: (1) glucose binding and enzyme-catalyzed oxidation resulting in the reduced form of enzyme; (2 A) oxidation of reduced form of enzyme by oxidized form of electron shuttle resulting in reduced form of electron shuttle; (3 A) transport of electrons to electrode via tethered or dissolved electron shuttles; (4 A) oxidation of reduced form of electron shuttles at working electrode; (2B) oxidation of reduced form of enzyme by
  • Sensor response data as a function of randomly ramped glucose concentration corresponding to a microneedle equipped with two high surface area Pt electrodes in contact with a redox hydrogel layer and glucose flux regulating polymer membrane top coating.
  • FIG. 21 Sensor response data as a function of linearly ramped glucose concentration corresponding to a custom chip equipped with two small-cross-section, high-surface-area Pt electrodes in contact with a redox hydrogel layer and a glucose-flux- regulating, polymer-membrane top coating.
  • Sensor operated in 2-electrode configuration i-t mode using +50 mV operating potential under ambient conditions with 100 mM PB buffer (pH 7.4) electrolyte.
  • FIG. 22 Low glucose concentration range sensor response data as a function of linearly ramped glucose concentration corresponding to a microneedle equipped with two high surface area Pt electrodes in contact with a redox hydrogel layer and glucose flux regulating polymer membrane top coating.
  • Sensor operated in 2-electrode configuration i- t mode using +50 mV operating potential under ambient conditions with lOO-mM PB buffer (pH 7.4) electrolyte.
  • Inset Corresponding calibration curve consisting of average steady state current values (averaged over the latter 100 seconds of each 200 sec sample interval) plotted as a function of glucose concentration (gray diamonds) and the corresponding linear fit (dashed line).
  • FIG. 23 Overlaid i-t traces corresponding to the response of a sensing electrode composed of a Pt wire coated with Gen. I cationic GOx hydrogel operating in hydrogen peroxide oxidation detection to various glucose concentrations. Sensor operated in 3- electrode configuration i-t mode with hydrogel coated Pt wire working electrode, Pt wire coil counter electrode and Ag/AgCl reference electrode operating at +600 mV under ambient conditions with 100 mM PB buffer (pH 7.4) electrolyte.
  • One aspect of the invention includes polymers.
  • the polymers are preferably configured to form hydrogels that can be used, for example, in next generation enzymatic biosensing technologies.
  • Polymers of the invention encompass polymers that include subunits of Formula I:
  • R 1 in each instance is independently H, alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof;
  • R 2 in each instance is independently N or O;
  • R 3 in each instance is independently:
  • R 4 in each instance is a spacer arm
  • R 5 in each instance is Formula II or Formula III:
  • R 6 , R 7 , and R 8 in each instance are independently H; alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof; a nitrogen protecting group; a tethered polypeptide; a tethered redox mediator; a linking arm; or a tethered subunit of Formula I, with the proviso that at least one of R 6 , R 7 , and R 8 may be absent; and
  • n in each instance is independently a positive integer.
  • alkyls described herein include substituted or unsubstituted, linear or branched, saturated carbon groups.
  • exemplary alkyls include (Cl-Cl8)alkyl, (Cl- Cl2)alkyl, and (Cl-C6)alkyl, such as (Cl)alkyl (methyl), (C2)alkyl (ethyl), (C3)alkyl (propyl, including p-propyl and isopropyl), and (C4)alkyl (butyl, including isobutyl, sec- butyl, and ieri-butyl).
  • cycloalkyls described herein include substituted or unsubstituted saturated cyclic alkyl groups.
  • exemplary cycloalkyls include (Cl-Cl8)cycloalkyl, (Cl- Cl2)cycloalkyl, (Cl-C7)cycloalkyl, or (Cl-C7)cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
  • the aryls described herein include substituted or unsubstituted functional groups containing one or more aromatic rings.
  • the aryls can be monocyclic, bicyclic, tricyclic, etc.
  • Exemplary aryls include phenyl, benzyl, naphthyl, anthracenyl, thienyl, and indolyl.
  • heterocycles and heteroaryls described herein include cycloalkyls and aryls in which one or more carbons are replaced with an atom other than carbon (e.g., sulfur, oxygen, or nitrogen).
  • substituents include alkyl, alkynyl, cycloalkynyl, cycloalkyl, hydroxyl, halo (e.g., Cl, Br, F), haloalkyl, aryl, aldehyde, halogen-substituted aryl (including aryl groups substituted multiple times with the same or different halogen), nitro-substituted aryl, heterocycle, heteroaryl, hydroxyl, halo, haloalkyl, amino, an amino protected with a nitrogen protecting group, nitro, pyridine, ester, amide, azide, sulfate, sulfite, sulfoalkyl, sulfhydryl, sulfonamide, thiazole, nitro, cyano, alkoxy, carboxy, aldehyde, a saturated or unsaturated cycloalkyl, a saturated or unsaturated heterocycle, bridged saturated and unsaturated c
  • nitrogen protecting groups described herein include benzyl ethers, silyl ethers, esters including sulfonic acid esters, carbonates, sulfates, and sulfonates, among others.
  • suitable nitrogen protecting groups include substituted methyl ethers; substituted ethyl ethers; -chlorophenyl, -methoxyphenyl, 2,4-dinitrophenyl, benzyl; substituted benzyl ethers (p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2- and 4-picolyl, diphenylmethyl, 5-dibenzosuberyl, triphenylmethyl, p-methoxyphenyl- diphenylmethyl, di(p-methoxyphenyl)
  • Polymers in which R 5 is Formula III can be made by including diacrylate or diacrylamide monomers, such as those shown in IFGS. 5 A and 5B, in the polymerization process.
  • n used herein for Formula I and Formula III refers independently in each instance to any positive integer. Examples include positive integers within a range of 1- 50,000 inclusive or more, such as 1-25,000, 1-10,000, 1-5,000, 1-2,500, 1-1,000, 1- 500, 1-250, 1-100, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, or 1-2.
  • the moiety of Formula II can be positively charged or neutral, depending on whether R 8 is present or absent.
  • the polymers described herein encompass both salt and non-salt forms.
  • the tethered polypeptide can include any polypeptide.
  • the polypeptide can have any number of amino acid residues, such as from 2 to about 10, to about 50, to about 100, to about 150, to about 300, to about 1000, to about 2,000, to about 3,000, to about 4,000, to about 4,500 or more residues.
  • the polypeptide can have any function.
  • the polypeptide can have a binding function, a structural function, an enzymatic function, or any other function.
  • the polypeptide is an enzyme.
  • exemplary types of enzymes include transferases, hydrolases, lyases, isomerases, and ligases.
  • Transferases are enzymes that transfer functional groups (e.g., amino or phosphate groups).
  • Hydrolases are enzymes that transfer water or catalyze the hydrolysis of a substrate.
  • Lyases are enzymes that add or remove the elements of water, ammonia, or carbon dioxide to or from double bonds. Ligases join two molecules.
  • the enzyme is preferably an oxidoreductase.
  • Oxidoreductases are enzymes that catalyze the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor.
  • Oxidoreductases usually utilize NADP, NAD+, FAD/FADH2 as cofactors.
  • Exemplary oxidoreductases include those falling under EC 1.1, which include oxidoreductases that act on the CH-OH group of donors (alcohol oxidoreductases); EC
  • EC 1.3 which include oxidoreductases that act on the CH-CH group of donors (CH-CH oxidoreductases); EC 1.4, which include oxidoreductases that act on the CH-NfL group of donors (amino acid oxidoreductases, monoamine oxidase); EC 1.5, which include oxidoreductases that act on CH-NH group of donors; EC 1.6, which include oxidoreductases that act on NADH or NADPH; EC 1.7, which include oxidoreductases that act on other nitrogenous compounds as donors; EC 1.8, which include oxidoreductases that act on a sulfur group of donors; EC 1.9, which include oxidoreductases that act on a heme group of donors; EC 1.10, which include oxidoreductases that act on diphenols and related substances as donors; EC 1.11, which include oxidoreductases that act on peroxide as an acceptor (
  • glucose oxidase GOx
  • notatin EC number 1.1.3.4
  • Glucose oxidase is an oxido-reductase that catalyzes the oxidation of glucose to hydrogen peroxide and D-glucono-5-lactone.
  • the tethered redox mediator comprises any compound or moiety capable of undergoing a reversible oxidation-reduction (redox) reaction, e.g., a reaction that involves a transfer of one or more electrons between chemical species.
  • redox mediators include those provided in FIGS. 5A-5I, 6, 7A, 7B, 8, and 18A-18C and analogs thereof.
  • Some redox mediators oxidize reduced molecules, such as a reduced oxidoreductase enzyme, and transfer the electrons to a medium, other molecules, or an electrode.
  • Such redox mediators are referred to herein as“electron shuttles.” Examples of electron shuttles are those provided in FIGS.
  • Electron shuttles can be identified by exhibiting transfer of electrons, e.g. electron transfer from reduced form of glucose oxidase to the oxidized form of an electron shuttle, in an inert atmosphere, e.g., in the absence of oxygen, such that hydrogen peroxide cannot be generated.
  • the electron shuttle comprises a compound of Formula VI:
  • R 12 and R 13 are each independently H, alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof; or are fused in an aromatic or non-aromatic ring;
  • R 14 is an electron withdrawing group, such as a sulfonate, a sulfonamide, an ammonium, a quaternary ammonium, a fluoroaklyl, a perfluoroalkyl, a nitro, a cyano, or a combination thereof;
  • R 15 is H, alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof;
  • the compound of Formula VI is optionally tethered to a monomer, polymer, or polymer subunit via any one or more of R 12 , R 13 , R 14 , R 15 , and any atom in the aromatic or non-aromatic ring formed by R 12 and R 13 .
  • R 12 and R 13 are fused in a C6 aromatic ring, wherein the C6 aromatic ring includes R 12 , R 13 , the carbons in Formula VI to which R 12 and R 13 are bound, and two additional carbons.
  • R 14 in the electron shuttle of Formula VI is Formula VII or Formula VIII:
  • R 16 is H, alkyl, alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, perfluoroalkyl, or a combination thereof;
  • R 17 is H, alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof; a tethered polymer; a tethered monomer; or a linking arm.
  • R 17 can be tethered to a polymer monomer or a polymer via any one of R 6 , R 7 , and R 8 as described for the polymers and monomers herein.
  • Analogs of the redox mediators explicitly provided herein include isomers and substituted versions of the redox mediators.
  • analogs of naphthoquinone include unsubstituted 1 ,4-naphthoquinone, unsubstituted 1, 2-naphthoquinone, unsubstituted 2, 3 -naphthoquinone, unsubstituted 2,6-naphthoquinone, and substituted versions thereof, including 2-hydroxy- 1, 4-naphthoquinone, 5-hydroxy- 1,4- naphthoquinone, 6-hydroxy- 1, 4-naphthoquinone, 3-hydroxy- 1, 2-naphthoquinone, 4- hydroxy- 1 ,2-naphthoquinone, 5-hydroxy- 1 ,2-naphthoquinone, 6-hydroxy- 1 ,2- naphthoquinone, 7-hydroxy- 1, 2-naphthoquinone, 8
  • redox mediators include sulfonate- or sulfonamide-substituted redox mediators, such as 1,2- naphthoquinone-4-sulfonates, l,2-naphthoquinone-4-sulfonamides, and others.
  • tethered redox mediator comprises a 1 ,2- naphthoquinone. See FIGS. 6-8.
  • the general term“1, 2-naphthoquinone” encompasses unsubstituted 1, 2-naphthoquinone (see“base structure” in FIG.
  • any substituted versions thereof such as 3-hydroxy- 1, 2-naphthoquinone, 4-hydroxy- 1, 2-naphthoquinone, 5-hydroxy- 1 ,2-naphthoquinone, 6-hydroxy- 1 ,2-naphthoquinone, 7-hydroxy- 1 ,2- naphthoquinone, 8-hydroxy-l, 2-naphthoquinone, and others.
  • Preferred 1,2- naphthoquinones of the invention comprise an electron withdrawing group at the 4- carbon of the 1, 2-naphthoquinone. (For the carbon numbering of l,2-napthoquinones, see the“base structure” in FIG.
  • Exemplary electron withdrawing groups include sulfonate, sulfonamide, ammonium, quaternary ammonium, fluoroaklyl, perfluoroalkyl, nitro, and cyano groups. Others known the art are acceptable.
  • the 1 ,2-naphthoquinones can be tethered to the polymer via the 3-carbon, the 4-carbon, the 5-carbon, the 6-carbon, the 7- carbon, or the 8-carbon of the 1,2- naphthoquinone. If tethered via the 4-carbon, the electron withdrawing group can serve as an intermediary between the tether and the 4- carbon.
  • the tethered subunits include tethered subunits having a structure of Formula I. Some tethered subunits having a structure of Formula I can be tethered via the nitrogen of Formula II at any one of R 6 , R 7 , or R 8 of the tethered subunit. Some tethered subunits having a structure of Formula I can be tethered via a nitrogen (derived from an amino) or sulfur (derived from a sulfhydryl) at R 9 of Formula IV. Accordingly, some polymers of the invention include subunits of Formula I tethered to each other via R 6 , R 7 , or R 8 of the respective tethered subunits.
  • Some polymers of the invention include subunits of Formula I tethered to each other via a nitrogen (derived from an amino) or sulfur (derived from a sulfhydryl) at R 9 of Formula IV in the respective tethered subunits.
  • Some polymers of the invention include subunits of Formula I tethered via R 6 , R 7 , or R 8 to a nitrogen (derived from an amino) or a sulfur (derived from a sulfhydryl) at R 9 of Formula IV in corresponding tethered subunits.
  • the corresponding tethered subunits can be from the same individual polymer backbone, thereby forming an intra-backbone crosslink, or can be from separate polymer backbones, thereby forming inter-backbone crosslinks.
  • the tethers thereby provide effective crosslinks between one or more individual polymer backbones in the polymer.
  • spacer arm refers to any linear, branched, and/or cyclic moiety connecting two other moieties.
  • the spacer arms in some aspects are preferably flexible.
  • the spacer arms can include substituted or unsubstituted C1-C25 alkylenes.
  • Exemplary spacer arms include one or more instances of a moiety selected from the group consisting of -(CH 2 ) m -, -(CH 2 ) m -0-(CH 2 ) m -, -(CH 2 ) m -(NR 18 R 19 )-(CH 2 ) m -, and combinations thereof.
  • R 18 and R 19 in a given spacer arm can independently be H; alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof; or a nitrogen protecting group, with the proviso that at least one of R 18 and R 19 may be absent.
  • the spacer arm can include moieties such as (-(CH 2 ) 2 -0) m -(CH 2 ) 2 -.
  • the subscript m in any given spacer arm is a positive integer. Examples include positive integers from 1 to 20, inclusive, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any ranges therebetween.
  • Tethers, tethering agents, and tethering arms used to tether two components to each other preferably include at least one linking group.
  • the term“linking group” refers to a moiety comprising a functional group capable of covalently reacting with (or reacted with) a functional group on another moiety. Moieties that are capable of interacting with each other are referred to herein as“corresponding linking groups.”
  • Preferred tethers, tethering agents, and tethering arms include one or more internal spacer arms, two or more terminal linking groups, and, optionally, one or more internal linking groups.
  • the spacer arms can include any spacer arm as described herein.
  • the terminal linking groups include functional groups capable of reacting with (or reacted with) linking groups on the components that are to be (or are) linked.
  • the internal linking groups are pairs of linking groups reacted within the tethers, tethering agents, and tethering arms themselves and can link two or more spacer arms to each other.
  • Tethers include at least two reacted linking groups at each end of the tether; tethering agents include at least two unreacted terminal linking groups; and tethering arms include at least one reacted linking group at one end of the tethering arm and at least one unreacted terminal linking group.
  • tethers actively link two components to each other via the reacted linking groups
  • tethering agents have the ability to link two components to each other via the unreacted terminal linking groups
  • tethering arms are linked to a first component via the reacted linking group and have the ability to link the first component to a second component via the unreacted terminal linking group.
  • Exemplary corresponding linking groups include those shown in FIGS. 11A-11C. Components to be tethered can originally contain these groups or can be modified to contain them.
  • At least one linking group on the tether, tethering agent, or tethering arm includes an amine-reactive functional group.
  • the amine-reactive functional group can be a primary amine-reactive functional group.
  • Amines can be included, for example, in the polymers at R 5 , at the N-terminus of polypeptide chains, and in the side- chain of lysine (Lys, K) amino acid residues. There are numerous synthetic chemical groups that will form chemical bonds with primary amines.
  • isothiocyanates include isothiocyanates, isocyanates, acyl azides, N-hydroxysuccinimide (NHS) esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, alkyl halides, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. Exemplary structures of some of these groups are shown in FIG. 10. Most of these conjugate to amines by either acylation or alkylation. In the case of alkylation, the carbon bound to the nitrogen is considered a reacted linking group (by virtue of an alkyl halide, for example, reacting with the nitrogen).
  • Formaldehyde and glutaraldehyde are aggressive carbonyl (-CHO) reagents that condense amines via amine-carbonyl condensation reactions, Mannich reactions and/or reductive amination.
  • NHS esters and imidoesters are common amine- specific functional groups that are incorporated into reagents for protein crosslinking and labeling.
  • the tether, tethering agent, or tethering arm can include a sulfhydryl (thiol) -reactive functional group as a linking group.
  • Sulfhydryls can be included, for example, in the side-chain of cysteine (Cys, C) amino acid residues.
  • Exemplary sulfhydryl-reactive functional groups include haloacetyl (iodoacetyl, bromoacetyl, etc.), maleimide, and pyridyldithiol groups.
  • Exemplary tethering agents are shown in FIGS. 9A-9D.
  • Various other suitable tethering agents or components thereof include any of the linkers or functional groups provided in the Thermo Scientific Crosslinking Technical Handbook (tools.thermofisher.com/content/sfs/brochures/ 1602163 -Crosslinking-Reagents- Handbook.pdf), which is incorporated herein by reference.
  • the polymer in some versions can take the form of cross-linked polymer networks that include individual polymer backbones cross-linked to each other.
  • the individual polymer backbones include the substituted alkylene chains (and terminal end groups) resulting from the polymerization of vinyl (ethenyl) groups in acrylic monomers.
  • the cross-links can take the form of tethers between individual polymer backbones.
  • the tethers can be formed post-polymerization via orthogonal chemistries on the polymer backbone by way of the pendant groups bearing reactive functionality. Suitable tethering agents used to form the tethers include those described above, particularly those having amine-reactive functional groups.
  • Cross-linked polymers comprising tethers between individual polymer backbones include polymers that include subunits of Formula I wherein R 5 is Formula II, wherein R 8 a tethered subunit having a structure of Formula I wherein R 5 in the tethered subunit has a structure of Formula II and is tethered at R 8 of the tethered subunit.
  • cross-links can also or alternatively take the form of cross-linkers polymerized into the individual polymer backbones.
  • Such cross-linkers preferably include an internal spacer arm and two or more terminal vinyl (ethenyl) groups.
  • Exemplary cross linkers are shown in FIGS. 1D, 3F, and 3G.
  • cross-linkers include polyethylene glycol dimethacrylates, l,6-hexanediol diacrylate, l,6-hexanediol dimethacrylate, l,9-nonanediol dimethacrylate, 1 ,4-butanediol dimethacrylate, 1,3- butanediol dimethacrylate, l,lO-decanediol dimethacrylate, diurethane dimethacrylate, l,4-butanediol diacrylate, ethylene glycol diacrylate, l,5-pentanediol dimethacrylate, 1,4- phenylene diacrylate, allyl methacrylate, 2,2-bis[4-(2-hydroxy-3- methacryloxypropoxy)phenyl]propane, tricyclodecane dimethanol diacrylate, tetraethylene glycol diacrylate, bis(2-methacryloxye
  • R 5 in at least one subunit of the polymer is Formula II
  • at least one of R 6 , R 7 , and R 8 in Formula II is Formula IV:
  • R 9 in each instance is independently hydrogen, alkyl, alkynyl, cycloalkyl, aryl, halogen- substituted aryl (including aryl groups substituted multiple times with the same or different halogen), nitro-substituted aryl, heterocycle, heteroaryl, hydroxyl, halo, haloalkyl, amino, an amino protected with a nitrogen protecting group, pyridine, ester, amide, azide, sulfate, sulfite, sulfoalkyl, sulfhydryl, sulfonamide, thiazole, nitro, cyano, alkoxy, carboxy, aldehyde, a saturated or unsaturated cycloalkyl, a saturated or unsaturated heterocycle, bridged saturated and unsaturated cylcoalkyl, fused aromatic, aromatic heterocycle, an N-hydroxysuccinimide ester-reactive group, an amine-reactive group, a hal
  • the backbone or cross-linked backbones in the polymer can include homopolymer backbones and/or copolymer backbones.
  • Copolymer backbones can include random co- polymers and/or block co-polymers.
  • Each backbone in the polymer can have from 1 to 50,000 subunits inclusive, such as from 1 to 25,000 subunits, 1 to 10,000 subunits, 1 to 5,000 subunits, 1 to 2,500 subunits, 1 to 1,000 subunits, 1 to 500 subunits, 1 to 250 subunits, 1 to 100 subunits, 1 to 50 subunits, 1 to 40 subunits, 1 to 30 subunits, 1 to 20 subunits, 1 to 10 subunits, 1 to 5 subunits, or 1 to 2 subunits.
  • the polymers of the invention can also include subunits of Formula V : wherein, in each instance of Formula V, Ri is H, alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof; n is a positive integer; and R 10 is carboxyl or carboxamido.
  • Such subunits can result from polymerizing acrylic monomers such as acrylamide, methacrylamide, acrylate, methacrylate, and analogs thereof and can be polymerized along with the acrylic monomer building blocks giving rise to the subunits Formula I.
  • the polymer can include the subunits of Formula I and Formula V in any relative proportion.
  • Exemplary proportions instance ratios of Formula I and Formula V of from about 1:1000 (Formula FFormula V) to about 1000:1 (Formula FFormula V), from about 1:500 (Formula FFormula V) to about 500:1 (Formula FFormula V), from about 1:100 (Formula FFormula V) to about 100:1 (Formula FFormula V), from about 1:1000 (Formula FFormula V) to about 1000:1 (Formula FFormula V), from about 1:1000 (Formula FFormula V) to about 2:1 (Formula FFormula V), from about 1:500 (Formula FFormula V) to about 1:1 (Formula FFormula V),.
  • Each instance of Formula I and Formula V is counted separately regardless of whether or not the instances of Formula I or Formula V can be grouped as contiguous blocks (/. ⁇ ? ., structures of Formula I or Formula V with n > 1).
  • the polymers of the invention can form hydrogels when sufficiently interlinked or interconnected and dispersed in water.
  • the hydrogels preferably include at least a polymer of the invention and water.
  • the hydrogel can include untethered redox mediators (including electron shuttles and those that oxidize/reduce hydrogen peroxide), salts, electrolytes, buffers, and other reagents or compounds dissolved or dispersed within the hydrogel.
  • the untethered components of the hydrogel are not tethered to the polymer and can diffuse freely therein.
  • the hydrogel can be included in an electrochemical cell.
  • the electrochemical cell has at least a counter electrode and a working electrode.
  • the hydrogel composition contacts, at the minimum, the working electrode.
  • An example of a suitable electrochemical cell includes a standard, three-electrode configuration that includes a working electrode, a counter electrode, and a reference electrode such that all electrodes, working and counter electrodes, or only the working electrode is equipped (in contact) with the hydrogel.
  • Other electrochemical cells can be used, including those with fewer electrodes such as a two-electrode electrochemical cell, which includes a counter electrode and a working electrode.
  • Working and counter electrode composition can include gold, platinum, or conductive carbon, among other materials.
  • the reference electrode can include silver, among other materials.
  • a preferred working and counter electrode composition is nanostructured platinum.
  • the electrochemical cells can be used to detect and/or determine the concentration of an analyte in a sample.
  • the sample can include a bodily fluid.
  • the bodily fluid can include interstitial fluid, intravascular fluid, lymphatic fluid, or transcellular fluid.
  • Particular examples of bodily fluids include amniotic fluid, aqueous humour, vitreous humour, bile, blood, blood plasma, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chime, endolymph, perilymph, exudates, feces, diarrhea, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), serous fluid, semen, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal
  • the analyte can include sugars, lipids, proteins, and small molecules, among others.
  • the analyte is preferably one that is capable of being oxidized, such that electrons resulting from its oxidation can be detected and quantitated.
  • a preferred analyte is glucose.
  • the electrochemical cells can be employed in a number of detection modes. These detection modes include direct hydrogen peroxide detection, electron shuttle-based detection, mediated hydrogen peroxide oxidation detection, mediated hydrogen peroxide reduction detection, and hybrids thereof. See, e.g., FIGS. 16A-16B, 17A-17D, and 19 and the following examples, which explain these detection modes in further detail.
  • the systems can include any component described herein in any combination.
  • Such components include polymers, enzymes, redox mediators, tethers, tethering arms, electrodes, etc.
  • a subset of the components, such as the polymers, enzymes, redox mediators, tethers, etc. can be provided in the form of a hydrogel.
  • Another aspect of the invention includes methods of detecting analytes.
  • the methods comprise contacting a sample containing the analyte with a system as described herein.
  • the system employed in the detection method comprises a tethered polypeptide, such as an oxidoreductase, and the detecting includes the oxidoreductase oxidizing the analyte.
  • the system employed in the detection method comprises a tethered redox mediator, and the detecting includes the redox mediator undergoing a redox reaction.
  • the system employed in the detection method comprises a polymer provided in the form of a hydrogel and an electrode in contact with the hydrogel, and the detecting includes the electrode undergoing a change in electric charge.
  • the analyte includes glucose.
  • Another aspect of the invention includes monomer building blocks useful for making the polymers described above, particularly monomer building blocks suitable for giving rise to the subunits Formula I in the polymers.
  • Such monomers include compounds having a structure of compound 4 or compound 5 or a salt thereof, wherein:
  • R 1 and R 8 in each instance are independently H, alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof;
  • R 2 in each instance is independently N or O;
  • R 3 in each instance is independently: H; alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof; or a nitrogen protecting group when R 2 is N; or
  • R 4 is a spacer arm
  • R 6 is H, alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof; or R 7 ;
  • R 9 is in each instance independently alkyl, alkynyl, cycloalkyl, hydroxyl, halo (e.g., Cl, Br, F), haloalkyl, aryl, aldehyde, halogen- substituted aryl (including aryl groups substituted multiple times with the same or different halogen), nitro-substituted aryl, heterocycle, heteroaryl, hydroxyl, halo, haloalkyl, amino, an amino protected with a nitrogen protecting group, nitro, pyridine, ester, amide, azide, sulfate, sulfite, sulfoalkyl, sulfhydryl, sulfonamide, thiazole, nitro, cyano, alkoxy, carboxy, aldehyde, a saturated or unsaturated cycloalkyl, a saturated or unsaturated heterocycle, bridged saturated and unsaturated cylcoalkyl, fused aromatic
  • R 1 , R 3 , R 6 , R 8 , and R 9 can each independently be substituted or unsubstituted methyl, ethyl, propyl, butyl, propyl, or hexyl.
  • at least one of R 6 and R 7 in compound 4 is substituted or unsubstituted methyl, ethyl, propyl, butyl, propyl, or hexyl.
  • at least one of R 6 , R 7 , and R 8 in compound 5 is substituted or unsubstituted methyl, ethyl, propyl, butyl, propyl, or hexyl.
  • the spacer arm of R 4 comprises one or more instances of a moiety selected from the group consisting of -(CH2) m -, -(CH2) m -0- (CH2) m -, -(CH2) m -(NR 18 R 19 )-(CH2) m -, and combinations thereof.
  • R 18 and R 19 in each instance can independently be H; alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof; or a nitrogen protecting group, with the proviso that at least one of R 18 and R 19 may be absent.
  • Each instance of m can independently be 1-20.
  • Another aspect of the invention includes methods of making compounds, such as the monomer building blocks described above.
  • An exemplary method includes one or more steps selected from the group consisting of:
  • R 1 and R 6 in each instance are independently H, alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof;
  • R 2 in each instance is independently N or O;
  • R 3 in each instance is independently:
  • R 4 is a spacer arm
  • R 7 is:
  • R 9 is in each instance independently alkyl, alkynyl, cycloalkyl, hydroxyl, halo (e.g., Cl, Br, F), haloalkyl, aryl, aldehyde, halogen- substituted aryl (including aryl groups substituted multiple times with the same or different halogen), nitro-substituted aryl, heterocycle, heteroaryl, hydroxyl, halo, haloalkyl, amino, an amino protected with a nitrogen protecting group, nitro, pyridine, ester, amide, azide, sulfate, sulfite, sulfoalkyl, sulfhydryl, sulfonamide, thiazole, nitro, cyano, alkoxy, carboxy, aldehyde, a saturated or unsaturated cycloalkyl, a saturated or unsaturated heterocycle, bridged saturated and unsaturated cylcoalkyl, fused aromatic
  • R 1 , R 2 , R 3 , R 4 , R 6 , and n in compound 4 are as defined above for compounds 1, 2, and 3, with the proviso that R 6 in compound 4 can be R 7 when R 6 in compounds 2 and 3 is H; and
  • R 8 is H, alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof;
  • X is a leaving group
  • R 1 , R 2 , R 3 , R 4 , R 6 , R 7 and n in compound 5 are as defined above for compound 4.
  • Steps a)-c) are preferably performed under inert atmosphere.
  • Step a) can be performed in an organic, nonpolar solvent such as chloroform (CHCI 3 ) or others.
  • suitable solvents include benzene, toluene, l,4-dioxane, and dichloromethane, among others.
  • Step b) can be performed in two sub-steps.
  • a first sub-step can involve reaction with a hydride in a polar protic solvent.
  • Suitable hydrides include sodium cyanoborohydride (NaBH 3 CN) and sodium triacetoxyborohydride (NaBH(OCOCH ) ), among others.
  • Suitable solvents include n-butanol, isopropanol, nitromethane, methanol, and ethanol, among others.
  • a second step can involve addition of a reagent such as acetic acid.
  • Step c) can be performed in a polar aprotic solvent such as tetrahydrofuran (THF).
  • a polar aprotic solvent such as tetrahydrofuran (THF).
  • suitable solvents include N- m eth y 1 p y rro 1 i do ne , ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, and propylene carbonate, among others.
  • the leaving group in compound 7 is preferably an anionic leaving group, such as halides (e.g., Cl , Br , and G) and sulfonate esters such as tosylate (TsO ). Other leaving groups are
  • An ion exchange step can be performed after step c) to provide the monomer with a desired counterion for downstream applications.
  • a preferred counterion for downstream applications is the chloride (Cl ) ion.
  • R 9 in R 7 , compound 6, and Formula IV can be ascertained from Cheung et al. (Cheung CW, Hu X. Nat Commun. 2016 Aug l2;7: 12494), Russianwieser et al. (Schrittwieser JH, Velikogne S, Kroutila W. Adv. Synth. Catal. 2015, 357, 1655-1685), Maya et al. (Maya, RJ, Poulose S, John J, Varma, RL. Adv. Synth. Catal. 2017, 359, 1177-1184), Moormann (Moormann A. Synthetic Communications. 1993, 23(6), 789- 795), and Ramachandran et al. (Ramachandran PV, Gagare PD, Sakavuyi K, Clark P. Tetrahedron Letters. 2010, 51, 3167-3169).
  • Additional monomers of the invention include monomers that are pre functionalized to contain a tethering arm, such as those provided in FIGS. 2A and 2B.
  • Another aspect of the invention is directed to the redox mediators, including the electron shuttles provided by Formulas VI, VII, and VIII, whether tethered to a polymer, tethered to a monomer, tethered to any other component provided herein, or provided in isolation.
  • Another aspect of the invention is directed to methods of making the systems provided herein.
  • the methods comprise polymerizing monomers to generate a polymer.
  • the monomers can include any one or more monomers provided herein.
  • the polymers can include any one or more polymers provided herein.
  • the monomers comprise terminal amines such as those shown in FIGS. 1A-1C and 3A-3G.
  • Such monomers can form subunits of a polymer after polymerization and can be tethered to other linear polymer chains, enzymes, and/or redox mediators.
  • the subunits can be tethered with the use of tethering agents having two unreacted terminal linking groups.
  • the subunits can also or alternatively be tethered with the use of linear polymer chains, enzymes, and/or redox mediators pre-functionalized with tethering arms comprising a unreacted terminal linking group. Tethering each component can occur simultaneously or sequentially. The tethering can occur with the same or different linking groups.
  • the monomers are pre-functionalized with tethering arms prior to polymerization, such as those shown in FIGS. 2A and 2B.
  • the monomers can include monomers having the same terminal linking groups or different terminal linking groups.
  • the monomers with different terminal linking groups can be polymerized in various specific proportions with respect to each other. Once polymerized, the monomers form polymer subunits having different terminal linking groups can provide specificity for tethering specific proportions of other components (e.g., enzymes, redox mediators, etc.) depending on the corresponding linking group present on each of the other components. Tethering components can occur simultaneously or sequentially.
  • Tethering any first component of the invention e.g., enzyme, redox mediator, tethering agent, tethering arm, first monomer, first polymer etc.
  • a any second component of the invention e.g., enzyme, redox mediator, tethering agent, tethering arm, second monomer, second polymer, etc.
  • Some versions include tethering a first component with a tethering arm to a second component lacking a tethering arm by linking the tethering arm of the first component to the second component.
  • Some versions include tethering a first component lacking a tethering arm to a second component with a tethering arm by linking the tethering arm of the second component to the first component. Some versions include tethering a first component with a tethering arm to a second component with a tethering arm by linking the terminal linking groups of the tethering arms to each other (provided the terminal linking groups on the tethering arms are corresponding linking groups). Some versions include tethering a first component lacking a tethering arm to a second component lacking a tethering arm by reacting each with a tethering agent having two unreacted terminal linking groups.
  • Some versions include tethering a first component with a tethering arm to a second component with a tethering arm by reacting each with a tethering agent having two unreacted terminal linking groups that correspond to the unreacted terminal linking groups on the tethering ar s.
  • aspects of the invention include any component described herein (monomers, polymers, cross-linked polymers, redox mediators, electron shuttles, enzymes, tethers, tethering agents, tethering arms, etc.), whether provided in isolation or in combination with the other components.
  • variable not explicitly defined in any particular structure or drawing herein e.g. ⁇ -R 19 , x, n, s, p, etc.
  • variable can be defined as in any other particular structure or drawing in which the variable is defined unless the context dictates otherwise.
  • Each instance of the same variable appearing more than once in any given structure is independent of the other instances (e.g. , can be different moieties defined for the variable) unless the context dictates otherwise.
  • Numerical ranges as used herein are intended to include every number and subset of numbers contained within that range, whether specifically disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, from 5 to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.
  • This example describes features, structures, and synthetic preparation of acrylamide and methacrylamide (and related analogs) monomer building blocks.
  • Some of the monomers are equipped with pendant oligo(ethylene glycol) spacer arms bearing terminal nitrogen-containing functional groups (FIGS. 3A-3C). Others have amines and alkylene spacer arms (FIGS. 3D and 3E).
  • the water-soluble monomers are designed and synthesized as building blocks of hydrophilic polymer-based functional materials, namely hydrogels for next generation enzymatic biosensing technologies.
  • the monomers can be prepared in very few synthetic steps using mild conditions and can be readily equipped with a wide range of functional groups.
  • Functionalized acrylamide and methacrylamide monomers disclosed herein, and their corresponding synthetic routes, provide multi-functional hydrogel materials for enzymatic biosensing.
  • These materials include networks of hydrophilic polymer backbones as well as the ability to immobilize enzymes; to tune mechanical properties, water content, and mass transport properties within the hydrogel materials; and to install active components bound within the polymer network.
  • the monomers are composed of acrylamide, alkylacrylamide (e.g., methacrylamide), acrylate, alkylacrylate (e.g., methacrylate), and/or related moieties that yield hydrophilic polymer backbones with, e.g., poly (ethylene glycol) (PEG) or other spacer arm-containing pendants bearing terminal amine or ammonium functional groups.
  • PEG poly (ethylene glycol)
  • the terminal amine or ammonium functional groups can subsequently act as sites for orthogonal chemistries or other additional functionalities.
  • FIGS. 1A-1D and 3A-3G illustrate molecular structures of some exemplary monomers of the invention. These include primary or secondary amine-functionalized monomers (FIGS. 1A, 3A, 3D, and 3E) that serve as both functional components for preparation of hydrogel materials and precursors for tertiary amine monomers (FIGS. 1B and 3B) and cationic quaternary ammonium monomers (FIGS. 1C and 3C).
  • the monomers as shown in FIGS. 1A-1C and 3A-3C can be generated with via a three-step synthetic route as illustrated below in Scheme 1. Each step results in a monomer that serves as the precursor for the next.
  • the synthetic route accesses all three types of monomers in three steps, affords access to an extremely wide range of analogs with an equally broad scope of functional groups positioned at the pendant group terminals, requires no protecting group chemistry, employs mild conditions, requires minimal purification, and provides building blocks of next generation hydrogel targets.
  • An additional fourth step can be employed to interchange the counterions of quaternary ammonium bearing monomers, e.g. a compound 5 monomer (scheme 1 illustrated below) for which iodide is the counterion can be converted to a compound 5 monomer for which chloride is the counterion by way of chloride form ion exchange resins.
  • R 1 , R 3 , R 6 , and R 8 are each independently H, alkyl, cycloalkyl, aryl, heterocycle, heteroaryl, or a combination thereof, except that R 6 in compound 4 can be R 7 when R 6 in compounds 2 and 3 is H.
  • R 9 in each instance is independently alkyl, alkynyl, cycloalkyl, hydroxyl, halo (e.g., Cl, Br, F), haloalkyl, aryl, aldehyde, halogen-substituted aryl (including aryl groups substituted multiple times with the same or different halogen), nitro-substituted aryl, heterocycle, heteroaryl, hydroxyl, halo, haloalkyl, amino, an amino protected with a nitrogen protecting group, nitro, pyridine, ester, amide, azide, sulfate, sulfite, sulfoalkyl, sulfhydryl, sulfonamide, thiazole, nitro, cyano, alkoxy, carboxy, aldehyde, a saturated or unsaturated cycloalkyl, a saturated or unsaturated heterocycle, bridged saturated and unsaturated cylcoalkyl,
  • the initial synthetic step is the reaction of acrylic and/or methacrylic anhydrides with an excess of diamine (Scheme 1, compounds 1 and 2, respectively) to yield the primary amine-functionalized monomer (Scheme 1, compound 3).
  • Compound 3 is then converted to tertiary amine compound 4 via a Borch type reductive ami nation that proceeds by a mechanism that efficiently alkylates the amine group but cannot proceed further to form any quaternary ammonium.
  • utilizing the mild reductive ami nation conditions yields an optimal pathway to cationic compound 5 that avoids the harsh reaction conditions and problematic purifications typically associated with direct conversion of primary amines to quaternary ammoniums.
  • the resulting reaction solution was allowed to slowly warm to room temperature (as the ice bath melted) and stirred overnight. Upon completion of the allotted reaction time the solvent was removed via roto-vap to afford the crude as a clear oil.
  • the crude material was purified via silica gel column chromatography using a ClUCUMeOthEtsN (18:3:0.5 by volume) solvent system and afforded the monomer of compound 3 as a light brown viscous oil in 58% yield.
  • the reaction for the preparation of compound 4 is preferably not completely sealed. This prevents pressure buildup due to gas formation.
  • the initial stage ran under inert atmosphere is preferably conducted under dynamic N2, and the second stage (during which the most gas is formed) is preferably performed with the septum removed, leaving the flask unsealed.
  • the reaction is preferably run in a properly functioning fume hood. Additionally, the roto-vap vacuum pump exhaust is preferably routed into fume hood as well.
  • the reducing agent NaCNBH 3 is toxic and can liberate very toxic gas if it comes in contact with acids. Special care is preferably taken to prevent any contact with the user. Special care is also preferably taken to prevent contact with acids and to prevent storage and handling in the presence of incompatible materials.
  • Example procedure for exchange of counterions of an exemplary monomer of compound 5 In order to exchange the counterion of an exemplary monomer of compound 5 from iodide counterions to chloride counterions Amberlite IRA-400 chloride form ion exchange resin (-20-40 g) was first loaded into an Erlenmeyer flask followed by -250 mL of Milli-Q water and washed by swirling, allowing the resin beads to settle to the bottom of the flask, and decanting the water from the flask.
  • the freshly washed resin was then loaded into a glass chromatography column, further washed by passing -200 mL of Milli-Q water through the column, activated by passing -200 mL of 1 M HC1 (aq.) through the resin containing column, and rinsed by passing -300-400 mL of Milli-Q water though the column.
  • the solvent was then switched to methanol by first draining the water from the column followed by successively passing -100-150 mL aliquots of methanol through the column.
  • Additional monomers of the invention include amine-containing monomers that are modified to contain a tethering arm prior to polymerization. See, e.g. , FIGS. 2A-2B.
  • Acrylate-based and alkylacrylate monomers corresponding to the acrylamide and alkylacrylamide moieties described herein are also encompassed by the invention and can be generated according to similar methods.
  • Acrylamide, alkylacrylamide, acrylate, alkylacrylate, and their functionalized analogs are generally water soluble, are among the very best monomers for hydrogel formation, and can be polymerized via several types of polymerizations including free radical polymerization and reversible addition-fragmentation chain-transfer polymerization (RAFT).
  • RAFT reversible addition-fragmentation chain-transfer polymerization
  • the resulting three-dimensional networks nearly always exhibit hydrogel properties due to the hydrophilicity of their polymer chains.
  • Preparation of substituted and/or functionalized acrylamides and methacrylamides can be achieved in good yields via several routes starting from cost effective and commercially available materials.
  • the monomers of the invention described herein have many favorable features: 1) acrylamide/methacrylamide moieties bearing PEG pendants with terminal functionality maintain water solubility of the monomers (a property that can be lost or negatively impacted if alkyl chain pendants are employed); 2) hydrophilicity of the corresponding polymer hydrogel materials is not diminished as a result of pendant functionality; and 3) the availability of diaminated PEG“oligomers” of well-defined lengths lends ease and cost effectiveness to the synthetic preparation of monomers of this architecture.
  • acrylamide- and methacrylamide-type monomers permit exploiting the utility of nitrogen-containing functional groups (in this case amines) for both forming acrylamide moieties and for creating suitable monomer building blocks for multi functional hydrogel materials.
  • nitrogen-containing functional groups in this case amines
  • These hydrogels are equipped with both reactive sites, useful for immobilizing enzymes and tethering redox species, and ionic functional groups that can facilitate more favorable electrostatic interactions between the hydrogel network and the hosted enzymes. Improving the electrostatic environment of the enzyme serves to aid in stabilization and prevent phase separation.
  • Acrylamide/methacrylamide preparation based on the reaction of PEG diamines with aery lic/methacry lie anhydrides results in the target monomer framework, a reactive functional group on the pendant terminals, and flexible pendant chains.
  • the flexibility of the chains lends utility to the corresponding hydrogel materials by allowing the pendant functional groups increased range of motion (relative to such groups bound in close proximity to polymer backbones) to help promote favorable reactions and interactions.
  • NQSA-2 The procedure synthesizing NQSA-2 (see FIG. 8 for structure) was based on J. Am. Chem. Soc. 2004, 126, 1024-1025.
  • a lOOO-mL Schlenk flask was charged with 1,2- naphthoquinone-4-sulfonic acid sodium salt (1 eq., 6.36 g, 24.44 mmol) and tetrabutylammonium chloride (1 eq., 6.79 g, 24.44 mmol), equipped with a stir bar, sealed via septum, interfaced with a Schlenk manifold, and placed under inert atmosphere.
  • Trifluoromethanesulfonic anhydride triflic anhydride; 1 eq., 24.4 mL of 1 M soln in CH2CI2, 24.4 mmol
  • the reaction of triphenylphosphine oxide with trifluoromethanesulfonic anhydride to form triphenylphosphine ditriflate was first accompanied by a pink/purple coloration of the reaction solution followed by the formation of a fine white precipitate.
  • the proper order of addition would be to add the activated sulfonate to the piperazine solution.
  • the order was reversed in this case due to the presence of undissolved solids that rendered transfer of the activated sulfonate solution problematic; we acknowledge that the order of addition surely had a negative impact on the reaction yield.
  • the solution was concentrated by purging the headspace with nitrogen until the volume was reduced to -450-500 mL.
  • the solution was transferred to a lOOO-mL separatory funnel, -250 mL of 2 M HC1 (Aq.) and -150 mL D.I.
  • the aqueous phase was extracted 3X with CH2CI2 (-350 mL/wash), the combined organic phases were dried with MgS0 4 , and solvent was removed by rotary evaporation to afford the crude product as a red solid.
  • hydrogel materials made with monomers such as those described in Example 1 and, optionally, electron shuttles such as that described in Example 2.
  • the hydrogel materials are useful for next generation enzymatic biosensors.
  • the hydrogel materials can include redox hydrogel functional materials containing oxidoreductase and/or non-oxidoreductase enzymes useful for next generation electrochemical enzymatic biosensors.
  • the hydrogel materials can also or alternatively include non-redox hydrogel functional materials that contain oxidoreductase and/or non- oxidoreductase enzymes.
  • the materials are prepared from acrylamide, methacrylamide, acrylate, and/or methacrylate monomers with pendant functional groups (such as those described in Example 1) either alone or in combination with non-functionalized acrylamide, methacrylamide, acrylate, and/or methacrylate co-monomers.
  • the pendant functional groups can include, for example, amine groups, cationic functional groups, and/or other functional groups, such as linking groups attached via amino groups.
  • the resulting materials include hydrophilic polymeric materials decorated with reactive sites and functional groups that can be modified for a wide range of specific applications.
  • the materials can facilitate signal transduction in sensing systems by serving to immobilize enzyme components, regulate mass transport rates of analytes and other dissolved compounds that participate in device function, and, in some versions, mediate transport of electrons from oxidoreductase enzymes to the sensing electrodes.
  • FIGS. 13A, 13C, 13E, and 13G illustrate structures of exemplary hydrogel materials (without tethered enzymes, which can be included).
  • Each combines key features of both solids and liquids (a feature of hydrogels), is readily tunable, affords stabilizing effects for biological enzyme catalyst components, can be readily formed in aqueous conditions compatible with enzyme components (/. ⁇ ? . , that preserve enzyme activity), and is amenable to a wide range of processing methods.
  • These multifunctional materials are well suited for next generation sensing technologies such as those designed for detection using very small sample volumes.
  • the functional hydrogel materials of the present invention include cross-linked copolymer networks prepared from acrylamide, methacrylamide, acrylate, and/or methacrylate co-monomers equipped with functional groups either alone or in combination with non-functionalized acrylamide, methacrylamide, acrylate, and/or methacrylate co-monomers that ultimately afford mechanisms to immobilize enzyme components, install tethered redox species, and tune a range of hydrogel properties.
  • the resulting hydrophilic polymer backbones can be equipped with pendants bearing terminal amine and ammonium functional groups (see, e.g., the structure shown in FIG. 13 A) and/or linking groups (e.g., those made with monomers as shown in FIGS. 2A-2B).
  • Amine groups are reactive sites for orthogonal chemistries that allow enzyme tethering and installation of functional components such as tethered redox mediators and enzymes (see, e.g., FIGS. 12A-12C, 13C, 13E, 13G, 14A-14B, and 15A-15Q).
  • Cationic ammonium groups participate in favorable electrostatic interactions with enzyme components that prevent phase separation (Heller, A.; Feldman, B. Accounts of Chemical Research 2010, 43, 963-973) and improve enzyme stability.
  • the elongated and flexible pendants facilitate favorable reactions and interactions by endowing amine and ammonium groups with increased amplitude of motion relative to such groups that are confined in close proximity to polymer backbones of the hydrogel network.
  • Hydrogels of the invention can be prepared from solutions of monomers, cross linker, enzyme, and photoinitiator in aqueous phosphate buffer (pH 7.4) by free radical polymerization using ultraviolet or visible light photocuring techniques (chemical curing, electropolymerization and other techniques are also viable).
  • the hydrogel forming solutions generally have overall monomer concentrations of 10-30 % (w/v), monomer mass ratios of 8:1:1 (acrylamide and/or methacrylamide and/or acrylate and/or acrylamide : amine monomer : cationic monomer), cross-linker concentrations of 1-5 % (w/v), and enzyme concentrations of 0.4-2.0 mg/mL.
  • Exemplary amine monomers are described above in Example 1 and shown in FIGS.
  • FIGS. 1A, 1B, 3A, 3B, 3D, and 3E Exemplary cationic monomers are described above in Example 1 and shown in FIGS. 1C and 3C. Exemplary monomers “pre-functionalized” with one of two members of corresponding linking groups are shown in FIGS. 2 A and 2B. Acrylate and methacrylate monomers corresponding to the acrylamide and methacrylamide monomers are encompassed by FIGS. 1A-1C and can be generated using procedures similar to those described above in Example 1. Any acrylamide-based monomer described herein can be replaced with a corresponding acrylate-based monomer. Exemplary cross-linking monomers are shown in FIGS. 1D, 3F, and 3G.
  • Water soluble photoinitiators employed for preparation of hydrogel materials include bis(mesitoyl)phosphinic acid sodium salt (Grutzmacher, H.; et al. Macromol. Rapid Commun. 2015, 36, 553-557) and 4,4’-azobis(4-cyanovaleric acid) (commercially available) at low millimolar concentrations typically in the range of 0.5-5 mM.
  • further modifications via orthogonal chemistries
  • can be performed post-polymerization see e.g., FIGS. 15A-15Q in order to tether enzymes or electron shuttles to the polymer backbones as well as cross-link the polymer backbones.
  • Enzyme tethering, redox mediator tethering, and polymer cross-linking can be performed in any of a number of different orders. See FIGS. 12A-12C. However, it is preferred that enzyme and electron shuttle tethering occurs prior to cross-linking.
  • Tethering of enzymes to the hydrogel network can be achieved by the coupling of reactive amine sites along the polymer backbones with amine- (e.g. , lysine) or thiol- (e.g., cysteine) bearing residues of the enzyme by treatment with bis- or multifunctionalized tethering agents as described herein.
  • amine- e.g. , lysine
  • thiol- e.g., cysteine bearing residues of the enzyme by treatment with bis- or multifunctionalized tethering agents as described herein.
  • FIGS. 9A-9D Exemplary tethering agents for enzyme tethering are shown in FIGS. 9A-9D and elsewhere herein. Schematic illustrating enzyme tethering are shown in FIGS. 12A-12C.
  • Analogous chemistries involving pendant amine groups can be employed to install tethered electron shuttles for versions of the invention employing redox polymer hydrogels (see, e.g., FIGS. 15A, 15E, 15F, 15L).
  • Electron shuttle redox species bearing carbonyl, epoxide, V-hydroxysuccinimide, and a variety of other functional groups subject to nucleophilic attack by amines (or other types of coupling reactions) result in covalent coupling and access a variety of redox hydrogels.
  • Exemplary electron shuttles that can be tethered within the hydrogel networks are shown in FIGS.
  • An exemplary enzyme that can be immobilized within the hydrogel networks is glucose oxidase (GOx), which can act as an oxidoreductase component.
  • GOx glucose oxidase
  • many other proteins or enzyme catalysts can be immobilized within the hydrogel networks.
  • Versions of the invention disclosed herein are designed to be most compatible with water- soluble enzymes such as GOx that are known to be polyanions at physiological pH (pH 7.4). Compatibility is achieved via favorable electrostatic interactions between the cationic pendants decorating the polymer backbones and the polyanionic enzyme. However, these materials can serve as effective scaffolds to immobilize polycationic and neutral enzymes as well.
  • Preparation of the functional hydrogels can be performed by first polymerizing monomer building blocks in the absence of cross-linking agents to yield soluble linear copolymer. The soluble linear polymer can be subsequently functionalized and cross- linked to form hydrogels.
  • Formation of the enzymatic redox hydrogel post-polymerization can include each of three distinct processes: (1) Covalent tethering of electron shuttle species to polymer through pendant functional groups; (2) Covalent tethering of enzyme to polymer through pendant functional groups (ultimately resulting in immobilized enzyme); and (3) Covalently cross-linking polymer chains into a polymer redox hydrogel equipped with tethered electron shuttle and immobilized enzyme. These steps can be performed simultaneously (FIG. 12A, panel C, and FIG. 12B), or separately (e.g. , sequentially) (FIG. 12C). If performed separately, the steps can be performed in any order. However, it preferred that electron shuttle tethering and enzyme tethering precede cross-linking.
  • electron shuttle tethering can precede enzyme tethering
  • enzyme tethering can precede electron shuttle tethering
  • electron shuttle tethering and enzyme tethering can occur simultaneously.
  • Employing multiple orthogonal chemistries constitutes a means to control one or more of the processes while the others remain inert.
  • both electron shuttle loading and degree of cross-linking can be precisely controlled, allowing for tuning and access to materials otherwise unattainable through a“single chemistry’” approach.
  • FIGS. 15A-15Q Examples of functionalizing the polymers post-polymerization with different orthogonal chemistries are shown in FIGS. 15A-15Q. These examples illustrate multiple orthogonal chemistry based strategies to independently install electron shuttle and cross link polymer chains to afford redox hydrogels.
  • an amine -bearing linear copolymer is equipped with a tethered electron shuttle via NHS-ester:amine coupling Chem. Soc. Rev., 2009, 38, 606-631) to yield an electron shuttle -bearing copolymer (FIG. 15A).
  • a second NHS-ester:amine coupling between the electron shuttle-bearing copolymer and a tetrazole compound (Angew. Chem. Int. Ed. 2008, 47, 2832 -2835) forms a tetrazole-bearing copolymer (FIG. 15C).
  • an amine-bearing linear copolymer is first equipped with tethered electron shuttle via copper-free click chemistry. This occurs by first installing azide functional groups (FIG. 15E) followed by treatment with electron shuttles bound to cyclooctyne groups such as DIMAC (Ellen M. Sletten and Carolyn R. Bertozzi. Org. Lett., 2008, 10(14):3097-3099) or ALO (Pamela V. Chang, Jennifer A. Prescher, Ellen M. Sletten, Jeremy M. Baskin, Isaac A. Miller, Nicholas J. Agard, Anderson Lo, and Carolyn R. Bertozzi. PNAS, February 2, 2010, vol. 107, no. 5, 1821-1826) (FIG.
  • the electron shuttle-bearing copolymer of FIG. 15F can be equipped with tetrazole groups (FIG. 151) and UV treated to induce tetrazole photo-click cross-linking (FIG. 15J) to yield a hydrogel structure.
  • Cross-linking can also occur using on epoxide linkers (FIG. 15K).
  • an amine-bearing linear copolymer undergoes carbodiimide driven coupling of a carboxylic acid-bearing electron shuttle with its pendant amine groups to yield electron shuttle-bearing copolymer (FIG. 15L).
  • the electron shuttle bearing copolymer can then undergo chemical cross-linking using NHS-ester bifunctional linkers (FIG. 15M).
  • NHS-ester functionalized diazirine and aryl azide compounds can be used for non-specific photochemical cross- linking with treatment with UV and near-UV radiation (Thermo Scientific Crosslinking Technical Handbook (tools.thermofisher.com/content/sfs/brochures/l602l63- Crosslinking-Reagents-Handbook.pdf)) (FIGS. 15N, 105, 105, and 105).
  • the cross- linking occurs by reactions of carbenes and nitrenes formed upon irradiation of diazirines and aryl azides, respectively (Thermo Scientific Crosslinking Technical Handbook, (tools.thermofisher.com/content/sfs/brochures/ 1602163 -Crosslinking-Reagents- Handbook.pdf)) (Angew. Chem. Int. Ed. 2009, 48, 6974 - 6998).
  • the electron shuttle analogs bearing complimentary linking groups used in the reactions of FIG. 15A, 10F, and 10L can be readily accessed using well-defined bioconjugation tools.
  • the electron shuttles used in the reactions of FIG. 15A and 15L can be prepared as shown in FIG. 15B using carbodiimide conjugation chemistry.
  • Electron shuttles bearing DIMAC and ALO cyclooctyne functional groups used in the reaction of FIG. 15F can be prepared using carbodiimide-driven carboxylic acid:amine coupling (FIGS. 15G and 15H). It is important to note that DIMAC and ALO are among the most water soluble of the cyclooctane compounds known to be effective copper free click coupling partners with azides and are therefore well suited for use in this system.
  • photoinduced cross-linking facilitates various solution processing strategies. Homogeneous hydrogel networks are readily accessed after solution processing via photocuring using wavelengths in the range of 254-350 nm UV ( Polym . Chem., 2014, 5, 2187-2201). Chemical cross-linking is also useful but can be more prone to inhomogeneity with respect to the spatial distribution of cross-links (caused by uneven mixing).
  • Enzyme immobilization steps can either be adapted or directly employed using the methods of FIGS. 15A-15Q and others.
  • monomer building blocks bearing different, non-amine terminal reactive functional groups e.g., linking groups
  • FIGS. 2A-2B monomer building blocks bearing different, non-amine terminal reactive functional groups
  • the different reactive functional groups facilitate independent cross-linking, installation of tethered electron shuttle, and/or immobilization of enzyme.
  • These monomers can be polymerized with or without the monomers bearing terminal amines (e.g., FIGS. 1A-1C and FIGS. 3A-3E).
  • the hydrogels of the invention serve several key purposes critical for use in sensing applications.
  • the hydrogels provide the abilities to tune and control mass transport of analytes within the hydrogel (such as concentration dependent glucose flux), to regulate the diffusion properties of participating water-soluble species, to regulate pH, and to stabilize enzyme components.
  • analytes within the hydrogel such as concentration dependent glucose flux
  • properties including mechanical strength, water content, pore size, and swelling/deswelling characteristics can be tuned.
  • Cross-linker structure namely the length and rigidity of the linker chain, as well as cross-linker mass ratio heavily influences pore size, which in turn influences mass transport factors such as analyte flux and the diffusion rates of ions and relevant water-soluble compounds within the hydrogels. Pore size also determines the degree of intermolecular interactions that govern (in part) enzyme stabilization.
  • the ability to tune and exploit mass transport properties within hydrogels, specifically the regulation of analyte flux into the hydrogel advantageously renders concentration-dependent analyte flux into the hydrogel the dominant factor governing electrochemical sensor response rather than the less stable activity of catalyst or electrocatalyst components (Heller, A. Annu. Rev. Biomed. Eng. 1999, 1, 153-175).
  • Such systems yield improved stability of the sensor response, which is advantageous considering the ever decreasing sample sizes of state of the art biosensor systems.
  • the multifunctional hydrogel materials disclosed herein address the materials- based needs of next generation electrochemical enzymatic sensing systems. Not only do the functional materials described herein meet the general safety and accuracy requirements for commercial electrochemical enzymatic sensing systems, but they also address the challenges associated with sensing using very small sample volumes.
  • the versatile hydrogel materials can be functionalized to facilitate a wide range of sensing mechanisms which makes them well suited to help minimize both the background signal caused by interferants and the susceptibility to sample-to-sample variances (such as variances in ambient oxygen concentration that can be challenging to address in glucose sensing systems); 2)
  • the tunability of the hydrogel system allows optimization of the stability and accuracy of sensor response through control of mass transport properties such as analyte flux and the diffusion rates of relevant dissolved species such as charge balancing ions; 3)
  • the combination of tunability and the presence of sites for orthogonal chemistry makes the hydrogels amenable to implementation of sensor amplification strategies such as redox cycling or indirect detection schemes (such as glucose detection based on differential oxygen); 4)
  • the materials afford a wide range of processing options granting the flexibility required to optimize factors such as electrode configuration and surface area for maximum sensor efficiency and sensitivity; and 5)
  • the system is generally versatile enough to address the majority of challenges
  • This example discusses an electrochemical glucose detection system based on functional hydrogel materials containing immobilized oxidoreductase enzymes, including those described in Example 3.
  • the general detection system architecture of the electrochemical glucose detection system includes working, counter, and reference electrodes functionalized with the enzyme-containing hydrogel material. Sensing is based upon direct or indirect measurement of electrons from enzymatic glucose oxidation. Signal transduction, in the form of an electrochemical response that is directly correlated with glucose concentration, is facilitated by the hydrogel material, which serves to immobilize the enzyme component (e.g., in close proximity of the electrodes), regulate mass transport rates of analytes (and other dissolved compounds that participate in device function), and, in redox hydrogel versions of the invention, mediate transport of electrons from the reduced enzyme to working electrodes.
  • FIGS. 16A and 16B show schematic representations of glucose sensing via direct hydrogen peroxide detection (FIG. 16A) and electron shuttle-based detection (FIG. 16B).
  • the hydrogel materials combine key features of both solids and liquids, are readily tunable, afford stabilizing effects for biological enzyme catalyst components, can be readily formed in aqueous conditions compatible with enzyme components (i.e. that preserve enzyme activity), and are amenable to a wide range of processing methods.
  • the ability to functionalize the hydrogel materials for device operation based on multiple detection modes affords the ability to tune operating voltages, maximize sensitivity, and minimize background signal.
  • the ability to tune and exploit mass transport properties within the hydrogels, specifically the regulation of glucose flux into the hydrogel generates a system in which concentration-dependent glucose flux into the hydrogel is the dominant factor governing electrochemical response rather than the less-stable activity of catalyst or electrocatalyst components.
  • the resulting glucose sensing electrodes are capable of glucose detection using sample volumes in the nanoliter/picoliter range, which is well below the present state of the art.
  • An exemplary oxidoreductase component of the detection system is glucose oxidase (GOx).
  • GOx glucose oxidase
  • the oxidized form of GOx selectively binds and oxidizes glucose to produce gluconolactone and the reduced form of GOx.
  • the reduced form of GOx can then undergo redox reactions with either oxygen to form hydrogen peroxide or with a suitable electron shuttle redox species, in both cases regenerating the oxidized form of GOx and transferring electrons from glucose oxidation to carriers that facilitate sensor signal transduction in the form of an electrochemical response.
  • the electrodes can be composed of a variety and/or combination of conductive materials and are preferably high surface area conductive materials.
  • Working and counter electrode materials can be gold, platinum, or conductive carbon (to name a few) while reference electrodes are silver.
  • Platinum electrodes with surfaces composed of platinum nanostructures/nanoparticles afford high electroactive surface area resulting in the increases in signal (relative to smooth platinum electrodes) for sensing using small sample volumes.
  • Nanostructured platinum electrodes are prepared from platinum electrodes using platinum electrodeposition methods. Electrodeposition of platinum nanostructures is achieved by a modified version of a reported procedure (Burke, J. J.; Buratto, S. K. J. Phys. Chem.
  • This deposition method results in, for example, ⁇ 60-fold increases in electrochemical surface area of electrodes covering a ⁇ l mm 2 area of a glass substrate relative to that prior to electrodeposition.
  • the large increases in electrochemical surface area afforded by the electrodeposition process is important for sensing using small sample volumes.
  • FIGS. 17A-17D are schematic cartoons depicting a device operating in each of these detection modes.
  • direct hydrogen peroxide detection mode FIG. 17A
  • the reduced form of GOx resulting from glucose oxidation FIG. 17A, 1
  • reduces oxygen FIG. 17A, 2)
  • hydrogen peroxide FIG. 17A, 3
  • the operating potential of this detection mode is defined by the potential at which electrochemical hydrogen peroxide oxidation occurs and is in the range of -400-700+ mV vs Ag/AgCl.
  • the electron shuttle detection mode allows device operation at lower potentials as this mode utilizes redox compounds (electron shuttles; e.g., FIGS. 5A-5I, 6, 7A, 7B, and 8) capable of oxidizing the reduced enzyme (FIG. 17B, 2B) (following glucose oxidation (FIG. 17B, 2B)), followed by diffusing of the reduced redox compounds to the working electrode (FIG. 17B, 3B) and oxidation at the working electrode (FIG. 17B, 4B) at lower voltage than electrochemical hydrogen peroxide oxidation.
  • Choice of electron shuttles allows tuning of operating potential for maximizing sensitivity and minimizing the background signal resulting from electrochemical reactions of other oxidizable species in the sample. Electron shuttle -based detection does not require the presence of oxygen.
  • Mediated hydrogen peroxide oxidation (FIG. 17C) and reduction modes (FIG. 17D) operate in a similar manner as direct hydrogen peroxide detection (FIG. 17 A) but can operate at lower positive (mediated hydrogen peroxide oxidation) or low negative (mediated hydrogen peroxide reduction) potentials by employing redox mediator compounds that oxidize (FIG. 17C, 4C) or reduce (FIG. 17D, 4D) hydrogen peroxide followed by their reformation at the working electrode (FIG. 17C, 5C; FIG. 17D, 5D).
  • redox mediator compounds that oxidize (FIG. 17C, 4C) or reduce (FIG. 17D, 4D) hydrogen peroxide followed by their reformation at the working electrode (FIG. 17C, 5C; FIG. 17D, 5D).
  • the electrochemical response corresponding to reformation the mediator species is proportional to glucose concentration.
  • mediator species include iron- or cobalt-based materials (FIGS.
  • FIG. 19 is a schematic representation of a dual detection mode version of the invention in which electrons from glucose oxidation are collected by way of both direct hydrogen peroxide oxidation and electron shuttle-based detection.
  • This dual detection mode employs redox polymers with tethered electron shuttles and/or non-redox polymers in hydrogels with untethered electron shuttles that can accept electrons from the reduced enzyme (resulting from glucose oxidation) and then be subsequently oxidized at the working electrode at potentials in a range overlapping that of electrochemical hydrogen peroxide oxidation.
  • electrons from glucose oxidation can be collected regardless of whether or not they participate in oxygen reduction to form hydrogen peroxide or in reduction of the electron shuttles.
  • the system can function independently of oxygen concentration as electrons from glucose oxidation can be measured regardless of their transport pathway.
  • This dual detection scheme is less susceptible to variations in environmental conditions (such as temperature, atmospheric pressure, atmospheric composition, etc.) that can influence the concentration of dissolved oxygen.
  • Redox cycling amplification amplifies the signal in very small sample volumes by exploiting electrodes and/or sacrificial electron donors for repeatedly replenishing the reduced product of the enzymatic process following initial oxidation of the of the reduced product, e.g. an electron shuttle, at the working electrode. This allows the reduced product to be repeatedly oxidized at the electrode. In this way, the number of electrons collected/measured at the electrode per molecule of analyte is multiplied, resulting in amplified signal.
  • the oxidized form of the enzyme is first reduced upon oxidation of the substrate, the reduced form of the enzyme is then oxidized by a precursor analog of the oxidized form of an electron shuttle (an analog that is not readily reduced by sacrificial donors) to form the reduced form of the electron shuttle, the reduced form of the electron shuttle is then oxidized at the electrode to form the oxidized form of the electron shuttle that can then undergo successive redox cycles of reduction by sacrificial electron donors followed by oxidation at the electrode.
  • the electrons originating from the substrate as well as the sacrificial donors can be collected, thereby amplifying the signal.
  • Some versions of the invention employ indirect glucose sensing based on differential oxygen.
  • the oxygen concentration is measured initially and as a function of time as sample glucose is oxidized and oxygen is consumed in the process.
  • Oxygen can be measured electrochemically in a very similar manner as pH (except that instead of the electrode being selective for protons it is selective for oxygen).
  • hydrogel-based electrochemical glucose sensing system exploits the versatility and tunability of custom multifunctional hydrogel materials to redefine the threshold for glucose sensing sample volumes.
  • This system meets the general safety and accuracy requirements for commercial electrochemical enzymatic glucose sensing systems and addresses the challenges associated with sensing using very small sample volumes.
  • the features of the hydrogel-based electrochemical glucose sensing system disclosed herein can be summarized as follows: 1)
  • the versatile hydrogel materials can be functionalized for device operation in multiple and even simultaneous detection modes, allowing background signal caused by interferants and susceptibility to variances in ambient oxygen concentration to be minimized; 2)
  • the tunability of the hydrogel system permits optimizing the stability and accuracy of sensor response through control of mass transport properties such as glucose flux and the diffusion rates of relevant dissolved species such as charge balancing ions; 3)
  • the combination of tunability and the presence of sites for orthogonal chemistry allows for the implementation of amplification strategies such as redox cycling or alternative detection modes such as those based on differential oxygen; 4)
  • the materials afford a wide range of processing options granting the flexibility required to optimize factors such as electrode configuration and surface area for maximum sensor efficiency and sensitivity; 5)
  • the system is versatile enough to address the majority of challenges associated with sensing using very small sample volumes; and 6) high surface area platinum electrodes with platinum nanostructure surfaces afford high electrochemical surface areas for sensing
  • Oxygen-free photoinitiator BAPO-Ona Macromol .
  • Redox hydrogels were prepared and installed onto device substrates by first weighing low-mg quantities (-5-15 mg) of the NQSA-2 electron shuttle (see Example 2) into a l.5-mL vial.
  • Linear copolymer solution (10 % w/v) was added in volumes such that the desired mole ratio of amine-bearing pendant groups to shuttle was achieved, and the resulting mixture was stirred for 3-5 minutes before glucose oxidase (GOx) enzyme (Calzyme) was added in the form of a 20-mg/mL stock solution in lOO-mM PB buffer (pH 7.4) to final concentrations of 1-5 mg/mL. The resulting mixture was stirred for 1-2 minutes.
  • the homobifunctional cross-linker sebacic acid bis(N-succinimidyl) ester was added to the desired mole ratio (relative to amine bearing pendant groups) in the form of a fine powder with stirring.
  • the resulting mixture quickly increased in viscosity as the reactions proceeded and was deposited onto device substrates (microneedles or chips with high surface area electrodes by drop casting, blade coating, or dip coating, depending upon the substrate.
  • the hydrogels were then allowed to cure overnight in the dark at high humidity. Once cured, the devices were soaked in buffer for several hours to swell the hydrogels and wash away leachables. The gels were then allowed to dry prior to installation of any glucose flux regulating membranes deposited via solution processing.
  • Solution-processed membranes were used for kinetic control (e.g., regulating glucose flux).
  • PEG polyurethane- linked polyethylene glycol
  • PDMS polydimethylsiloxane
  • M n 2500
  • These materials and their preparation can be found in the patent literature (US Patent 5,777,060 and US Patent 5,882,494).
  • Membranes were installed via drop casting, dip coating, or spray coating from 1-4% (w/w) solutions in THF, l,4-dioxane, or ethanol and allowed to dry in air at room temperature for 10-20 min.
  • Electrochemical glucose sensing with redox hydrogel-based sensors included either custom microneedles or glass chips with two high-surface-area platinum electrodes in contact with a layer of redox hydrogel and a polymer membrane top coating. Sensors were evaluated/operated in 2-electrode configuration i-t mode with sensing (working) electrodes poised at 50 mV relative to the counter electrode.
  • the employed sample solution/electrolyte was either 100 mM PB buffer (pH 7.4) or 50 mM PBS containing 154 mM NaCl (pH 7.4).
  • Sample volumes ranged from 50 pL (for chips only, sample in the form of droplet placed atop the electrode area) to 2-10 mL (microneedles with tips submerged into sample solution). Data shown here corresponds to sensors operated at ambient temperature in air. Glucose concentrations were varied randomly or linearly and sensors were typically operated for time intervals of 150-1000 sec for each glucose concentration, at which time data collection was paused and the sample solution was swapped for the next glucose concentration before resuming data collection. A typical sample swap took no longer than 30-60 sec. Results are shown in FIGS. 20-22.
  • Hydrogel forming solution was prepared by combining monomer stock solutions with 30% (w/v) concentrations in lOO-mM PB buffer (pH 7.4) to the desired weight ratio (ex: 7:3: 1, AA : MeAA-PEG-MH : MeAA-PEG-NMe3), adding cross-linker (tetra(ethylene glycol) diacrylate) to desired weight fraction (1-2 % w/v; added as pure material) and GOx enzyme to the desired concentration (0.4-3.0 mg/mL; added in the form of a 10-20 mg/mL stock solution in buffer), diluting with buffer to a final concentration of 10% (w/v), and degassing via sparging with nitrogen.
  • cross-linker tetra(ethylene glycol) diacrylate
  • the devices were soaked in buffer for several hours to swell the hydrogels and wash away leachables. The gels were then allowed to dry prior to installation of any glucose flux-regulating membranes deposited via solution processing. Data shown here corresponds to a Pt wire coated with GOx hydrogel without any flux regulating membrane layer.
  • Electrochemical glucose sensing with peroxide detection-based sensor Electrochemical glucose sensing with peroxide detection-based sensor.
  • GOx hydrogel-based electrochemical glucose sensors included either dip-coated Pt wire sensing electrodes, custom microneedles, or glass chips with two high-surface-area platinum electrodes in contact with a layer of GOx hydrogel with or without a polymer membrane top coating. Sensors were evaluated/operated in 2- or 3 -electrode configuration i-t mode with sensing (working) electrodes poised at 600 mV relative to the counter electrode.
  • the employed sample solution/electrolyte was either 100 mM PB buffer (pH 7.4) or 50 mM PBS containing 154 mM NaCl (pH 7.4).
  • Sample volumes ranged from 50 pL (for chips only, sample in the form of droplet placed atop the electrode area) to 2-10 mL (microneedles with tips submerged into sample solution). Data shown here corresponds to sensors operated at ambient temperature in air. Glucose concentrations were varied randomly or linearly and sensors were typically operated for time intervals of 150-1000 sec for each glucose concentration, at which time data collection was paused and the sample solution swapped for the next glucose concentration before resuming data collection. A typical sample swap took no longer than 30-60 sec. Alternatively, individual i-t traces were collected for each glucose concentration. Results are shown in FIG. 23.

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Abstract

L'invention porte sur des systèmes de détection électrochimique, des composants de ceux-ci, des matériaux et des procédés pour la fabrication des systèmes et des composants de ceux-ci et des procédés pour la détection d'analytes avec les systèmes. Les systèmes comprennent des cellules électrochimiques contenant des hydrogels. Les hydrogels comprennent des polymères portant des groupes pendants à fonction amine. Les polymères peuvent être réticulés par l'intermédiaire des groupes pendants à fonction amine. Les polymères peuvent également ou en variante comprendre des enzymes et/ou des navettes de transfert d'électrons qui y sont attachées par l'intermédiaire de groupes pendants à fonction amine. L'invention concerne également des monomères pour la fabrication des polymères. Les systèmes peuvent être utilisés pour détecter des analytes tels que le glucose.
PCT/US2018/063866 2017-12-04 2018-12-04 Systèmes de détection électrochimique et composants de ceux-ci Ceased WO2019113085A1 (fr)

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